We propose to bring to market, a novel regenerative medicine based therapy for fecal incontinence (FI). FI is the recurrent uncontrolled passage of fecal material. The Internal Anal Sphincter (IAS) is responsible for maintaining anorectal continence. Function of the IAS is dependent upon the neuromuscular integrity of both the smooth muscle and the intrinsic enteric nervous system (ENS). Currently, animal models of fecal incontinence do not mimic symptoms of passive FI observed in humans. We propose to further develop a novel surgical technique developed in lab to induce fecal incontinence in rabbits by damaging the IAS and utilize this model to validate the efficacy of implantation of bioengineered innervated IAS constructs for the treatment of FI. Our surgical procedure does not result in damage to the external anal sphincter (EAS), thereby limiting the injury to the smooth muscle layer of the IAS. Examination through anorectal manometry indicates the loss of IAS resting pressure as well as the neurally mediated rectoanal inhibitory reflex (RAIR) following partial IAS sphincterectomy. Our preliminary results indicate that rabbits undergoing partial IAS sphincterectomy are incontinent, with altered fecal behavior similar to symptoms of passive FI observed in humans (Aim 1). We have developed tissue culture based techniques to isolate circular smooth muscle cells from the IAS, and enteric neuronal progenitor cells from jejunal biopsies. We have optimized our bioengineering technique to generate innervated IAS constructs with neuromuscular integrity, including identifying factors important for neural progenitor cell differentiation. We propose to expand our bioengineering design to co-culture these two cell types to generate bioengineered autologous intrinsically innervated IAS constructs. These constructs are physiologically functional, and will be subject to intensive quality control to evaluate myogenic and neurogenic properties. We propose to conduct a pre-clinical randomized rabbit trial, whereby we will implant bioengineered IAS constructs in incontinent rabbits (Aim 2). The functionality of these constructs will be evaluated in vivo using anorectal manometry, and examine the reinstatement of the RAIR. Our preliminary studies indicate that reinstatement of this neurally mediated reflex occurs following implantation of bioengineered constructs. Lastly, in order to move this project towards an Investigative New Drug (IND) application to the FDA and qualify for First-in-Human studies, we propose to streamline our manufacturing and quality control protocols to fit Current Good Manufacturing Processes (CGMP) guidelines (Aim3). Our grant proposal is tailored to directly remedy the underlying pathology of FI, namely the loss of the neuromuscular integrity of the IAS. Our preliminary results provide proof of concept for the success of this regenerative medicine approach for treatment of FI.